Lysosomes are a critical component of eukaryotic cells, playing a role in degradation and recycling processes, signal transduction and extracellular secretion(I). Ion channels expressed on the endo-lysosomal membrane are crucial in intracellular signalling and maintaining the acidic luminal pH for optimal hydrolase activity(II). There are a number metabolic disorders, known as lysosomal storage diseases, that arise from lysosomal dysfunction(III).
Furthermore, targeting the autophagic-lysosomal pathway is a novel therapeutic strategy for clearance of toxic aggregates, which are pathological hallmarks of many neurodegenerative diseases. Endo-lysosomal channels have been historically challenging to investigate due to their intracellular location in small-sized organelles. However, advances in lysosomal biology have developed a technique to enlarge and extract endo-lysosomes to be recorded using conventional patch-clamp methods.
We applied a refined manual patch-clamp technique to characterize endogenous endo-lysosomal ion channels in their native environment, suitable for investigating potential therapeutic agents. In the present study we focused on the activity of TRPML and TMEM175 channels, due to their respective implications in mucolipidosis type IV(IV) and Parkinson’s disease(II). Moreover, we investigated how pH differences found along the endocytic pathway can affect TRPML channel activation.
We developed a high-throughput, electrophysiological assay of TREK-1 function to identify novel modulators. The assay was optimized to identify both activators and inhibitors, providing comprehensive mechanistic data for high value, limited supply screening libraries, such as the venom fraction library used in this study (Targeted Venom Discovery Array, T-VDA, Venomtech, UK).
Scientifica’s PatchScope Pro is an integrated electrophysiology rig incorporating an inverted phase-contrast fluorescence microscope, motorised XY stage and PatchStar micromanipulators, suitable for patch-clamp recording.